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Neuroblast senescence in the aged brain augments natural killer cell cytotoxicity leading to impaired neurogenesis and cognition

Nature Neuroscience volume 24, pages 61–73 (2021)Cite this article

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Abstract

Normal aging is accompanied by escalating systemic inflammation. Yet the potential impact of immune homeostasis on neurogenesis and cognitive decline during brain aging have not been previously addressed. Here we report that natural killer (NK) cells of the innate immune system reside in the dentate gyrus neurogenic niche of aged brains in humans and mice. In situ expansion of these cells contributes to their abundance, which dramatically exceeds that of other immune subsets. Neuroblasts within the aged dentate gyrus display a senescence-associated secretory phenotype and reinforce NK cell activities and surveillance functions, which result in NK cell elimination of aged neuroblasts. Genetic or antibody-mediated depletion of NK cells leads to sustained improvements in neurogenesis and cognitive function during normal aging. These results demonstrate that NK cell accumulation in the aging brain impairs neurogenesis, which may serve as a therapeutic target to improve cognition in the aged population.

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Fig. 1: Accumulation of NK cells in the dentate gyrus of normal aged human brains.
Fig. 2: NK cells in the brain and peripheral organs of young and aged mice.
Fig. 3: Single-cell profiling identifies increased activation and cytotoxicity of NK cells in the aged dentate gyrus.
Fig. 4: Fate mapping of NK cells reveals that local proliferation contributes to NK cell accumulation in the aged dentate gyrus.
Fig. 5: IL-27 derived from aged neuroblasts supports NK cells.
Fig. 6: Aged dentate gyrus neuroblasts express cellular senescence signatures and produce SASP factors.
Fig. 7: NK cells eliminate aged neuroblasts.
Fig. 8: Altered expression of MHC class I molecules in aged neuroblasts and effects of NK cell removal on cognitive function in aged mice.

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Data availability

Data were uploaded to Mendeley Dataset: https://doi.org/10.17632/bbzd4mwt8r.1. Bulk-RNA and single-cell RNA-sequencing data have been deposited in the NCBI Gene Expression Omnibus with the accession number GSE157772. Other information that supports the findings of this study is available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank H. Li, Y. Feng, Y. Li, X. Yang, M. Yuan, Y. Xiu, J. Fan, Y. Zhang and H. Zhao for technical assistance. We thank S. X. Shi for editorial assistance. This study was supported in part by the National Science Foundation of China (91949208, 91642205, 81830038, 81971094 and 81771274); the Advanced Innovation Center for Human Brain Protection, Capital Medical University, Beijing, China; and the National Key Research and Development Program of China (2018YFC1312200).

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Authors and Affiliations

  1. China National Clinical Research Center for Neurological Diseases, Jing-Jin Center for Neuroinflammation, Beijing Tiantan Hospital, Capital Medical University, Beijing, China

    Wei-Na Jin, Wenyan He & Fu-Dong Shi

  2. Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, Tianjin, China

    Wei-Na Jin, Kaibin Shi, Fu-Dong Shi & Qiang Liu

  3. School of Forensic Medicine, Shanxi Medical University, Jinzhong, China

    Jun-Hong Sun

  4. Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA

    Luc Van Kaer

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Contributions

Q.L. and F.-D.S. formulated the concept and designed the studies. W.-N.J., K.S., W.H. and J.-H.S. performed the experiments. W.-N.J., K.S. and Q.L. analyzed the results. Q.L., W.-N.J., L.V.K. and F.-D.S. interpreted the results. Q.L., W.-N.J., L.V.K. and F.-D.S. wrote and edited the manuscript.

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Correspondence to Qiang Liu.

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Peer review information Nature Neuroscience thanks Valery Krizhanovsky, Hongjun Song, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 NK cells reside within the parenchyma of aged human and mouse brains.

a, Immunofluorescence staining of aged human brain sections containing dentate gyrus shows that NK cells do not co-localize with endothelial cells (CD31+). Images are representative of 5 subjects (72 ± 6.7 years) with similar results. Scale bars: 40 µm (inset: 20 µm). b, Immunofluorescence staining of wild type male aged (18 months) mouse brain sections containing dentate gyrus shows that NK cells do not co-localize with endothelial cells (CD31+). Images are representative of 5 aged (18 months) mice. Scale bars: 40 µm (inset: 20 µm).

Extended Data Fig. 2 Single cell transcriptome analysis reveals increased NK cell activity in aged mouse brains.

CD45high immune cells were sorted by flow cytometry from spleen and dentate gyrus tissues of wild type male young (3 months) and aged (18 months) mice. Subsequently, single-cell RNA-sequencing was performed using the 10x Genomics Chromium platform. Unsupervised clustering and t-distributed stochastic neighbor embedding (tSNE) projections were performed on 6,422 cells from young spleen, 6,649 cells from young dentate gyrus, 5,521 cells from aged spleen, and 7,299 cells from aged dentate gyrus. a, Unsupervised hierarchical clustering was performed on CD45high cells obtained from aged spleen and aged dentate gyrus. Heatmap shows markedly altered gene expression in CD45high cells obtained from aged dentate gyrus versus aged spleen. b, Violin plots show the indicated immune cell subsets including NK cells in aged (18 months) dentate gyrus. c. Volcano plot shows that genes expressing NK cell signatures were enriched in the aged (18 months) dentate gyrus.

Extended Data Fig. 3 RNA sequencing reveals differentially expressed gene clusters in neuroblasts from young and aged dentate gyrus.

Single-cell suspensions were prepared from dissected dentate gyrus of male young (3 months) and aged (18 months) DCX-mRFP reporter mice. Thereafter, mRFP+ neuroblasts were sorted via flow cytometry. a, Heat map shows RNA transcriptomes of 1,101 differentially expressed genes from neuroblasts of young and aged dentate gyrus. The relative abundance of transcripts is indicated by the color (red, high; blue, low). b, Enrichment analysis was performed to measure the significance of altered gene expression by Gene Ontology annotations. The enrichment of pathways was analyzed by Fisher exact test. c, Real-time PCR analysis of selected genes that reflect neuroblast functions, such as cell growth and death, cell cycle, immune regulation, etc, in aged neuroblasts versus young neuroblasts. n = 3 per group. d, Bubble diagram displays enriched pathways based on function in the KEGG database. Pathway categories with FDR <0.05 were reported. e. KEGG database was used to build the network of differentially expressed genes of aged/young neuroblasts according to the relationship among mRNA, miRNA and proteins in the database. Error bars represent s.e.m.

Extended Data Fig. 4 Aged neuroblasts express SASP and cell cycle arrest genes.

a, Single-cell suspensions were prepared from dissected young (3 months) and aged (18 months) dentate gyrus tissues of male wild type mice. Thereafter, several cell types in the aged dentate gyrus were identified by flow cytometry using antibodies to surface markers. Quantitative RT-PCR analysis of SASP and cell cycle gene expression in isolated cell types from young and aged dentate gyrus, including neurons (CD15−CD29lowCD24hi), microglia (CD11b+CD45int), astrocytes (GLAST+), neural stem cells (GLAST+EGFR+CD24−) and neuroblasts (GLAST−EGFR−CD24low). Neuroblasts are a major cell population that possess the most upregulated genes among SASP components and cell cycle arrest genes (outlined in green), as well as upregulated Il27 gene (outlined in green). b, Bar graph shows quantification of IL-27 mRNA levels in cells from young and aged dentate gyrus. **p<0.01 by two-tailed unpaired Student’s t-test. p=0.0023, t=6.934, df=4. c, Immunostaining shows IL-27-expressing neuroblasts in aged (18 months) dentate gyrus, but not in young (3 months) dentate gyrus. Scale bar: 40 µm. n = 8 per group. **p<0.01 by two-tailed unpaired Student’s t-test. p=0.002, t=3.777, df=14. Error bars represent s.e.m.

Extended Data Fig. 5 Aged neuroblasts induce NK cell expansion in vitro.

Single-cell suspensions were prepared from dissected young (3 months) and aged (18 months) dentate gyrus tissues of male wild type mice. NK cells were harvested from spleen of young (3 months) mice. Co-culture experiments were performed using NK cells and FACS-sorted dentate gyrus cell types including neurons (CD15-CD29lowCD24hi), microglia (CD11b+CD45int), astrocytes (GLAST+), neural stem cells (GLAST+EGFR+ CD24−) and neuroblasts (GLAST−EGFR−CD24low). NK cell proliferation was assessed by measuring Ki67-expressing NK cells using flow cytometry. n = 4 per group. **p<0.01 by two-tailed unpaired Student’s t-test. p=0.0101, t=3.702, df=6. Error bars represent s.e.m.

Extended Data Fig. 6 Characterization of the senescence status of neuroblasts.

a, Immunostaining and quantification show expression of the senescence marker P16 in DCX+ cells of male young (3 months) and aged (18 months) mice. Scale bar: 20 µm. n = 6 per group. *p<0.05 by two-tailed unpaired Student’s t-test. p=0.0119, t=3.069, df=10. b, Immunostaining and quantification show BrdU incorporation in DCX+ cells of young and aged mice. Scale bar: 20 µm. n = 6 per group. *p<0.05 by two-tailed unpaired Student’s t-test. p=0.0103, t=3.153, df=10. c. The proportion of aged neuroblasts in the G1/G0, S and G2/M cell cycle phases of young and aged neuroblasts was measured by flow cytometry. n = 5 per group. *p<0.05 by two-tailed unpaired Student’s t-test. p=0.045, t=2.374, df=8. d, RNA-sequencing of aged neuroblasts reveals dysregulated expression of genes related to DNA damage response (DDR). e, KEGG database was used to build the network of differentially expressed DDR genes in aged (18 months) neuroblasts according to the relationship among mRNA, miRNA and proteins in the database. Data are representative of three independent experiments. Error bars represent s.e.m.

Extended Data Fig. 7 Senescence of aged neuroblasts increases NK cell number and activity.

Single-cell suspensions were prepared from dentate gyrus of male young (3 months) and aged (18 months) DCX-mRFP reporter mice. Thereafter, mRFP+ neuroblasts were sorted using flow cytometry and cultured in vitro. a, Real-time PCR and western blotting assessment of senescence markers (P16, P21 and P53) at mRNA and protein levels in cultured neuroblasts receiving treatment with mTOR inhibitor rapamycin or vehicle for 48 h. *p<0.05, **p<0.01 by two-tailed unpaired Student’s t-test. Left: n = 5 per group. P16: p=0.006, t=3.705, df=8; P21: p=0.0237, t=2.786, df=8; P53: p=0.0007, t=5.324, df=8. Right: n = 3 per group. P16: p=0.0324, t=3.217, df=4; P53: p=0.0073, t=5.037, df=4. b, Flow cytometry analysis of senescence-associated β-galactosidase (β-gal) expression in groups of cultured young (3 months) and aged (18 months) neuroblasts receiving indicated treatment for 48 h. n = 4 per group. **p<0.01 by one-way ANOVA. F2,9=20.01. Aged + vehicle vs. aged + rapamycin, p=0.002, q=7.012, df=9; aged + vehicle vs. aged + mTOR siRNA, p=0.0006, q=8.319, df=9. c, Flow cytometry analysis of IL-27 expression in groups of cultured young (3 months) and aged (18 months) neuroblasts receiving indicated treatment for 48 h. n = 8 per group. **p< 0.01 by one-way ANOVA. F2,21=11.0. Aged + vehicle vs. aged + rapamycin, p=0.0022, q=5.525, df=21; aged + vehicle vs. aged + mTOR siRNA, p=0.0011, q=5.976, df=21. d, Flow cytometry analysis of expression of NKG2D or Granzyme B in groups of NK cells after coculture with aged (18 months) neuroblasts receiving indicated treatment for 48 h. n = 8 per group. **p<0.01 by one-way ANOVA. NKG2D: F3,28=15.22. Aged + vehicle vs. aged + rapamycin, p=0.0045, q=5.276, df=28; aged + vehicle vs. aged + mTOR siRNA, p<0.001, q=7.413, df=28; aged + vehicle vs. aged + IL-27 siRNA, p<0.001, q=8.927, df=28. Granzyme B: F3,28=8.171. Aged + vehicle vs. aged + rapamycin, p=0.0094, q=4.865, df=28; aged + vehicle vs. aged + mTOR siRNA, p=0.0015, q=5.873, df=28; aged + vehicle vs. aged + IL-27 siRNA, p=0.001, q=6.106, df=28. Data are representative of three independent experiments. Error bars represent s.e.m.

Source data

Extended Data Fig. 8 Antibody depletion of NK cells in aged mice.

Anti-NK1.1 mAb (PK136) was used to deplete NK cells. 100 μg of anti-NK1.1 mAb was injected i.p. into each mouse every 5 days until the end of experiments. Single-cell suspensions were prepared from blood or brain tissues of male aged mice receiving anti-NK1.1 mAb or IgG control. a-b, Flow cytometry analysis of NK cell counts in blood (a) and brain (b) of aged (18 months) mice receiving anti-NK1.1 mAb or IgG control. n = 5 per group. **p<0.01 by two-tailed unpaired Student’s t-test. In a, p<0.0001, t=10.76, df=8. In b, p=0.0002, t=6.632, df=8. Data are representative of three independent experiments. Error bars represent s.e.m.

Extended Data Fig. 9 Absence of NKT cells does not affect neurogenesis and cognitive function in aged mice.

a, Immunostaining reveals the counts of Nestin+ neural stem cells and DCX+ neuroblasts in dentate gyrus tissues of male aged (18 months) wild type and CD1d-/- (devoid of NKT cells) mice. n = 6 per group. b, Spatial memory performance was measured in aged (18 months) wild type and CD1d-/- mice using Morris water maze. Bar graph shows swimming velocity in indicated groups of mice. n = 6 per group. c, Cumulative data plots show swimming distance of aged wild type and CD1d-/- mice. Swimming distance summary was from beginning to the end of each trial session from day 1 to 4. Images show swimming traces of indicated groups during the probe test at day 5. n = 14 per group. d-e, Quantification of time spent in the northeast quadrant during the probe test (d) and target-platform crossing times (e). In d-e, n = 14 mice per group. Data are representative of three independent experiments. Error bars represent s.e.m.

Extended Data Fig. 10 Reduced apoptosis of dentate gyrus neuroblasts in aged mice receiving anti-NK1.1-mAb.

a-b, Immunostaining (a) and quantification (b) show Caspase-3+DCX+cells in the aged (18 months) dentate gyrus of male wild type mice receiving one-month treatment of anti-NK1.1 mAb or IgG control. Scale bar: 40 µm. n = 6 mice per group. *p<0.05 by two-tailed unpaired Student’s t-test. p=0.0186, t=2.805, df=10. Data are representative of three independent experiments. Error bars represent s.e.m.

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Unprocessed western blots of Fig. 6e.

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Unprocessed western blots of Extended Fig. 7a.

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Jin, WN., Shi, K., He, W. et al. Neuroblast senescence in the aged brain augments natural killer cell cytotoxicity leading to impaired neurogenesis and cognition. Nat Neurosci 24, 61–73 (2021). https://doi.org/10.1038/s41593-020-00745-w

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